Interchain disulphide-bond formation in the assembly of immunoglobulin G. Heavy-chain dimer as an intermediate.
1. The role of disulphide-bond formation in the assembly of G(2a) myeloma protein 5563 was studied by pulse-labelling ascitic plasma cells of tumour-line 5563 for 2-8min. with radioactive amino acids, and analysing the intracellular proteins. Myeloma-protein determinants were first purified by ion-exchange chromatography under conditions that do not dissociate non-covalently linked sub-units of immunoglobulin G. The pulse-labelled material was then analysed by electrophoresis on polyacrylamide gels in sodium dodecyl sulphate-phosphate-urea buffer, which dissociates non-covalently linked sub-units; after gel electrophoresis, radioactive protein bands were located by radioautography, and characterized immunologically after elution. 2. Two heavy-chain intermediates were detected: (i) heavy-chain dimer; (ii) the dimer with one light chain attached. Free light chains had previously been shown to be intermediates in assembly. No evidence for the presence of half-molecules (one light chain attached to one heavy chain) was obtained. The formation of the disulphide bond between the heavy chains thus appears to precede the light-chain-heavy-chain linkage in immunoglobulin G assembly.
D000818:Animals; D001201:Ascites; D001345:Autoradiography; D002247:Carbon Isotopes; D002852:Chromatography, Ion Exchange; D004586:Electrophoresis; D007074:Immunoglobulin G / Q000096:biosynthesis*; D007930:Leucine / Q000378:metabolism; D051379:Mice; D009101:Multiple Myeloma / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000378:metabolism; D010950:Plasma Cells / Q000378:metabolism; D011506:Proteins / Q000032:analysis; D013440:Sulfides / Q000096:biosynthesis*; D014316:Tritium; D014633:Valine / Q000378:metabolism
D002247:Carbon Isotopes; D007074:Immunoglobulin G; D011506:Proteins; D013440:Sulfides; D014316:Tritium; D007930:Leucine; D014633:Valine